The objective of this project is to develop a safe, efficacious, single- dose, cost-effective vaccine for protection of humans against disease caused by Japanese encephalitis virus (JEV). A genetically defined, live attenuated vaccine will be developed by the introduction of specific mutations into a cDNA clone of JEV. The infectious clone system will be used to identify and introduce two other independent attenuating mutations into the JEV gene to reduce the risk of reversion. The resulting recombinant virus with triple attenuating amino acid substitutions, will be characterized biologically, with respect to stability, virulence, transmissibility and immunogenicity. Replication in cell cultures approved for manufacture of human products will be defined, and master and manufacturer's working seeds and pilot lots of vaccine prepared under GMP. Safety, immunogenicity and protection tests will be performed in monkeys. At the completion of the work to be funded under this proposal, it is anticipated that the vaccine candidate will be ready for initial clinical trials. PROPOSED COMMERCIAL APPLICATION: JE is a major health threat in Asia. Licenses inactivated vaccines are too expensive to produce and distribute (they require multiple doses) for routine use in most Asian countries. The vaccine developed in this program would be used for routine childhood immunization in Asia. The estimated market for a single dose childhood JE vaccine in Asia exceeds $400 MM/year. A substantial marked (>$100 MM/year) also exists for US and European travelers to Asia and for US Military.